During the last three decades this laboratory has studied the development of intestinal host defenses that protect neonates from infections and allergic diseases in the contaminated extrauterine environment. We have systematically defined host defense immaturities in animal and human models of small intestinal development. During the last ten years as part of a MERIT award, we have begun to determine the effect of breast milk on the maturation of these defined host defense immaturities using established human intestinal models of development (cell lines, organ cultures and xenograft transplants). As part of this approach we have begun to examine DNA microarrays of laser capture microdisection epithelial RNA from immature vs mature enterocytes, confirmed by qRT-PCR. The preliminary studies suggest that the immune response of immature enterocytes after exposure to exogenous (LPS) and endogenous (IL-12) inflammatory stimuli is excessive, e.g., a 20 fold increase in IL-8 in immature vs mature enterocytes. Using these techniques we have shown that enterocyte surface TLRs and their signaling molecules are upregulated which can account for the enhanced innate inflammatory response. In addition, the cellular inhibitors of TLR-induced inflammation (SIGIRR, IRAK-M, Tollip, A-20 etc) are underexpressed, another possible cause of excessive inflammation. Accordingly, our working hypothesis for this renewal reapplication is that human milk provides a protective link from mother to newborn in the extrauterine environment by actively stimulating the maturation of innate immune defense in immature enterocytes and thereby prevents the expression of age-related infectious and immune-mediated diseases during that period. Accordingly, to address this hypothesis, we will use human intestinal models and DNA microarrays to determine in experimental detail three representative anti-inflammatory factors in breast milk, e.g. 1) the regulatory cytokine, TGF-22; 2) the direct effect of a representative breast milk oligosaccharide, galacto-oligosaccharide and 3) the effect of Bifidobacteria and Lactobacillus, the predominantly colonizing bacteria in breast fed infants, media secretory products on the development of intestinal innate immunity in nursing newborns. These studies should help define the mechanism(s) of breast milk protection in newborns and support the importance of breast feeding and may help provide legislation to support working mothers breast feeding longer.